A Molecular Mechanism of Integrin Crosstalk: α_vβ₃ Suppression of Calcium/Calmodulin-dependent Protein Kinase II Regulates α₅β₁ Function

Human peripheral blood monocyte-derived macrophages were prepared as previously described (2). The human erythroleukemic cell line K562 transfected with cDNA encoding α_vβ₃ (Kα_vβ₃), α_vβ₃ in which the tyrosine residue at position 747 or 759 was mutated to phenylalanine (Kα_vβ₃Y747F and Kα_vβ₃Y759F, respectively) and Tacβ₃ (chimera of the extracellular and transmembrane domain of the IL2 receptor α-chain [Tac subunit] and the cytoplasmic tail of β₃, KTacβ₃) were derived and maintained as described (2, 3). In addition K562 cells were similarly transfected with cDNA encoding Tacβ₃ in which the serine residue at position 752 was replaced with proline (KTacβ₃S-P), cysteine (KTacβ₃S-C), alanine (KTacβ₃S-A), or glutamic acid (KTacβ₃S-E). Expression of all Tacβ₃S-X chimeras was equivalent to Tacβ₃ (3) as determined by flow cytometry as described (2; see Table I). For construction of Tacβ₃S-X, the HindIII and XhoI fragment of pTacβ₃ encoding the CT of β₃ (3) was ligated into HindIII-XhoI–digested pBluescript (Stratagene), creating pBSKSPB3TAIL. This contruct was subjected to PCR using a 5′ T7 oligonucleotide (Stratagene) with the 3′ oligonucleotide (5′-CCCCCCTCGAGTTAAGTGCCCCGGTACGTGATATTGGTGAAGGT-XXX-CGTGGC-3′) where XXX is AGG for S⁷⁵²P, ACA for S⁷⁵²C, GCT for S⁷⁵²A, and TTC for S⁷⁵²E. The resulting products were digested with HindIII-XhoI and ligated into pTacβ₃ digested with HindIII-XhoI, creating pTacβ₃S-P, pTacβ₃S-C, pTacβ₃S-A, and pTacβ₃S-E, respectively.

K562 cells also were transfected with full-length α_vβ₃ in which the serine at position 752 of β₃ was mutated to alanine as described for the mutation of β₃ tyrosine residues (4). In brief, nested PCR was performed on pBLY100 using the overlapping oligonucleotides 5′-GAGGCCACGCCTACCTTCACCAATATCACG-3′ and 5′-CTCCGGTGCGGATGGAAGTGGTTATAGTGC-3′ encoding the S-A mutation with oligonucleotides in the mutation cassette (4). After the nested PCR reaction, the wild-type β₃ CT was replaced with the S-A mutant CT by NdeI-NheI restriction. Transfection, selection, and fluorescent cell sorting for expression levels of α_vβ₃S752A equivalent to wild-type α_vβ₃ was as described previously, resulting in Kα_vβ₃S752A (Table I). Modified cDNAs were verified by dideoxy nucleotide sequencing.

Phagocytosis assays were performed as described (2) by flow cytometry using either FITC-FN– or FITC-mAb16 (anti-α₅)–coated 3.0-μm beads. Data are presented as a Phagocytic Index, the number of beads internalized per 100 cells.

Chemotaxis assays were performed in modified Boyden chambers (Neuroprobe) using 14.0-μM polycarbonate filters as described (19). Vitronectin (VN), fibronectin (FN), and BSA were added to basal chambers at 5 μg/ml and mAb at 10 μg/ml were added to apical chambers coincident with cells. Cells in Iscove's Modified Eagle's Medium (IMDM) adjusted to 1 mM Ca²⁺ and 1 mM Mg²⁺ with 0.5% human serum albumin and 2.0 mM Mn²⁺Cl were incubated for 4 h at 37°C in a humidified 5% CO₂ atmosphere for migration. Migration was quantitated by counting the number of cells per high power field on the underside of the filter after Giemsa staining.

Adhesion assays were performed as described in FN-coated (10 μg/ml) microtiter wells (2). Data are presented as the percent of added cells adherent after 1 h at 37°C.

Transfected K562 cells or monocyte-derived macrophages were stimulated as described in the text, washed once by centrifugation in ice-cold IMDM and suspended in ice-cold homgenization buffer containing Hepes (50 mM), EDTA (4 mM), EGTA (2 mM), sucrose (0.25 M), dithiothreitol (1 mM), phenylmethylsulfonyl fluoride (0.2 mM), Na₃VO₄ (2.0 mM), NaF (5.0 mM), phenyl-arsine-oxide (10.0 mM), and leupeptin (10 μg/ml), pH 7.5. Suspended cells were sonicated on ice and assayed for CamKII activity against the synthetic substrate autocamtide II (KKALRRQETVDAL) (21). An aliquot of cell extracts was used for protein determination by BCA. Parallel aliquots were assayed for CamKII activity in a 25-μl reaction mixture containing Hepes (50 mM), magnesium acetate (10 mM), Na₃VO₄ (2.0 mM), NaF (5.0 mM), phenyl-arsine-oxide (10.0 mM), CaCl₂ (1 mM), calmodulin (0.1 μM; Sigma Chemical Co.), autocamtide II (20 μM), and γ-[³²P]ATP (0.1 mM, 3,000 cpm/pmol). The reaction was initiated by ATP addition and terminated by addition of trichloroacetic acid to a final concentration of 10%. The reaction mixture was centrifuged through phosphocellulose separation units (Pierce) and washed as described (26). CamKII activation results from phosphorylation events that result in kinase activity which is no longer dependent upon exogenous calcium or calmodulin. CamKII activity in cellular extracts was measured by quantitating the incorporation of radioactive phosphate into a synthetic CamKII substrate (autocamtide-2) in the presence (calcium/calmodulin-independent + calcium/calmodulin-dependent activity) or absence (calcium/calmodulin-independent activity) of calcium and calmodulin. The activation of CamKII (autonomous activity) is expressed as a direct percentage of the total cellular CamKII activity (1) in which:

where autonomous activity equals the CamKII activity without calcium or calmodulin and total activity equals CamKII activity with calcium or calmodulin.

CamKII expression levels in transfected cell lines was assessed by immunoprecipitation as previously described, followed by Western blot analysis (1).

Kα_vβ₃, KTacβ₃, and KTacβ₅ were infected with a replication defective adenovirus in which the E1 region was replaced with the CMV early promoter and the cDNA for a constitutively active CamKII (AdCMV.CKIID3) or β-galactosidase (AdCMV.gal) and viral stocks propagated and titered as described (1). Transfected K562 cells at 5 × 10⁶/ml in IMDM were infected with recombinant adenovirus at a multiplicity of infection of 100 for 1 h followed by the addition of normal growth medium to dilute cells to a concentration of 0.5 × 10⁶/ml. After 4–6 h, cells were harvested for analysis of CamKII activity or functional assay as described in Results. Viability of all infected cell types exceeded 85% at the initiation, and 70% at the conclusion of experimental time courses.

FN was purified by gelatin affinity and VN by heparin affinity as previously described (2, 3). Monoclonal antibodies 7G2 (anti–human β₃), W6/ 32 (anti–human HLA), IC12 (anti–human α_v), AP3 (anti–human β₃), P1F6 (anti–human β₅), 4E3 (anti-IL2Rα, gp55, TAC), and mAb16 (anti– human α₅) have been previously described and were used in excess at 5.0 μg/ml unless otherwise indicated (2, 3).

The kinase inhibitors H7 (50 nM), KN62 (2.5 μM), KN04 (5.0 μM), KT5926 (20 nM), and ML-9 (2 μM) were included in some assays where indicated and all were from LC Laboratories (Woburn, MA). All other reagents were from Sigma Chemical Co. unless otherwise indicated.

Data are presented as the mean ± SEM from at least three replicates for all studies. Significance was determined by analysis of variance followed by Duncan's comparison testing. A minimum confidence interval of 95% was employed for all studies.

We have previously described a phenomenon, termed integrin crosstalk, in which ligation of α_vβ₃ prevents α₅β₁-mediated phagocytosis in macrophages and in K562 cells expressing transfected α_vβ₃. To determine if integrin crosstalk regulated α₅β₁ functions other than phagocytosis, we evaluated the effects of α_vβ₃ ligation on the migration of K562 cells on the α₅β₁ ligand FN. K562 cells did not migrate specifically to FN in IMDM containing 1 mM Ca²⁺ and 1 mM Mg²⁺. However, addition of 2 mM Mn²⁺ or the α₅β₁ conformation-stabilizing mAbs 8A2 or A1A5 at 5.0 μg/ml greatly enhanced the FN-specific migration of these cells, consistent with a requirement for high affinity α₅β₁ in migration (data not shown). As shown in Fig. 1 A, the migration of untransfected K562 to FN in the presence of 2 mM Mn²⁺ was enhanced sixfold over migration to the nonspecific protein casein; this migration was completely inhibited by mAb to α₅β₁ (data not shown). We also examined α_vβ₃-mediated migration in K562 expressing this transfected integrin in addition to the endogenous α₅β₁. Kα_vβ₃ migrated in response to VN (Fig. 1 A); migration response to VN was inhibited by mAb to α_v or β₃ (data not shown). However, migration of Kα_vβ₃ to FN was severely impaired compared with untransfected or mock transfected K562 (Fig. 1 A). Migration of Kα_vβ₃ to FN was restored by the addition of the ser/thr kinase inhibitor H7 (50 nM), while addition of H7 had no effect on Kα_vβ₃ migration to VN (data not shown). Restored migration of Kα_vβ₃ to FN in the presence of H7 was completely inhibited by mAb to β₁ (data not shown). These results completely parallel the previously described α_vβ₃-mediated crosstalk which inhibits α₅β₁-mediated phagocytosis (3) and support the hypothesis that the coligation of α_vβ₃ by FN regulates α₅β₁-mediated K562 cell migration to FN because this function, like phagocytosis, requires a high affinity form of α₅β₁.

To demonstrate definitively that α_vβ₃ regulation of α₅β₁-mediated migration was another example of integrin crosstalk, we examined migration to FN in KTacβ₃ and KTacβ₅, K562 cells expressing chimeric molecules comprised of the extracellular domain of the IL2 receptor and the cytoplasmic tail domain of the β₃ or β₅ integrin, respectively. Expression of Tacβ₃, but not Tacβ₅, leads to constitutive inhibition of α₅β₁-mediated phagocytosis in K562 cells (3; see Fig. 7 B). Expression of Tacβ₃, but not Tacβ₅ (Fig. 1 A) or Tac lacking a cytoplasmic tail (KTacNT, Fig. 2 A), completely inhibited α₅β₁-mediated migration to FN. The constitutive inhibition of migration to FN in KTacβ₃ was reversed by the addition of 50 nM H7 (Fig. 2 A). These studies demonstrate that α₅β₁-mediated migration and α₅β₁-mediated phagocytosis are similarly regulated by α_vβ₃ or the isolated β₃ CT and that this regulation is dependent upon a ser/thr kinase regulated by H7. These data suggest that both α₅β₁-mediated migration and α₅β₁-mediated phagocytosis are regulated by α_vβ₃-initiated crosstalk.

We have previously demonstrated that expression of the isolated β₃ cytoplasmic tail is sufficient for initiation of α_vβ₃ crosstalk (Fig. 1 A and reference 12). To further delineate the required sequence elements of this unique regulatory pathway, we introduced point mutations in the β₃ cytoplasmic tail and analyzed their effects upon α_vβ₃-initiated crosstalk to α₅β₁-mediated migration.

In a spontaneously occurring Glanzmann's Thrombasthenia mutation, the serine residue at position 752 of the β₃ CT is mutated to proline (6). This mutation results in loss of platelet β₃ function and a severe bleeding disorder. In vitro study has shown that Ser752 of the β₃ CT is required for the conformational change associated with elevated affinity of β₃ for ligand (8). To test whether Ser752 also is required for integrin crosstalk, we expressed an α_vβ₃ receptor in K562 cells in which Ser752 of β₃ was mutated to Ala (Fig. 1 B). While the ligation of wild-type α_vβ₃ blocked α₅β₁-mediated migration on FN (Fig. 1 B), the S752A mutant migrated as well as the untransfected cells. In addition, the S752A mutant migrated as well as wild-type α_vβ₃ on VN (Fig. 1 B), consistent with reports that this mutation does not affect ligand binding by β₃ integrins (8). This demonstrates that failure of the S752A mutant to initiate crosstalk did not result from an inability to recognize ligand.

Recently, a tyrosine in the β₃ cytoplasmic tail, Tyr747, has been implicated in activation-dependent α_vβ₃ adhesion to VN (4). In contrast to the S752A mutation, Y747F had no effect on α_vβ₃-initiated integrin crosstalk (Fig. 1 B). Consistent with the previous report of a requirement for this tyrosine in firm adhesion, the Y747F mutation did abolish migration of Kα_vβ₃Y747F to VN (Fig. 1 B). Mutation of Tyr759 to Phe (Y759F) did not affect either crosstalk or the migration function of α_vβ₃. These data demonstrate that the crosstalk signaling and adhesive functions of α_vβ₃ have distinct and independent sequence requirements in the β₃ cytoplasmic tail.

To evaluate further the requirement for β₃ S752 in integrin crosstalk, additional mutations at that position were made in the consititutively inhibitory Tacβ₃ construct. Mutation of Ser752 to Glu, Pro, or Cys as well as Ala abolished the inhibitory activity of Tacβ₃ on α₅β₁-dependent migration (Fig. 2 A) and α₅β₁-dependent phagocytosis (Fig. 2 B). Like the wild-type β₃ cytoplasmic tail, none of the mutants affected K562 binding to FN-coated surfaces, a function that does not require the high affinity state of α₅β₁ (Fig. 2 C). The addition of H7 reversed the Tacβ₃ inhibition of α₅β₁-mediated migration and phagocytosis (Fig. 2, A and B).

α_vβ₃ ligation inhibits the α₅β₁ high affinity functions of phagocytosis and migration, without effect upon α₅β₁-mediated adhesion. Alterations in α₅β₁ affinity can be regulated by calcineurin, a calcium/calmodulin-dependent phosphatase and CamKII (calcium/calmodulin-dependent protein kinase II; reference 1). Recently, inhibition of CamKII activity by α_vβ₃ ligation in smooth muscle cells was reported (1). Therefore, we evaluated α_vβ₃ regulation of CamKII as a potential mediator of α_vβ₃-initiated crosstalk.

CamKII activity was measured in human monocyte-derived macrophages in the presence and absence of an α₅β₁-specific phagocytosis target (mAb-16–coated latex beads) (3). Ligation of macrophage α₅β₁ with mAb-16 beads enhanced CamKII activity twofold, while ligation with a control target (W6/32 beads) had no effect (Fig. 3 A). Both basal and stimulated CamKII activities were decreased by the CamKII inhibitor KN62, but not the structurally related, but non-inhibitory KN04. Ligation of α_vβ₃ with soluble mAb 7G2 prevented the rise in CamKII activity induced by mAb-16 beads (Fig. 3 A). No additional decrease in CamKII activity was detected when KN62 and 7G2 were combined. Thus, β₃ ligation prevented the α₅β₁-induced rise in CamKII activity.

To explore further the hypothesis that CamKII mediates α_vβ₃ regulation of α₅β₁, we evaluated the regulation of CamKII in Kα_vβ₃. Binding of mAb-16 beads to Kα_vβ₃, and to vector-transfected K562 (data not shown), resulted in an increase in CamKII activity (Fig. 3 B) that was not seen when Kα_vβ₃ were incubated with W6/32 beads that bound to the cells equivalently. As in macrophages, the α₅β₁-mediated rise in CamKII activity was prevented by ligation of α_vβ₃ with soluble mAb 7G2 (Fig. 3 B) and by 7G2 Fab fragments or Arg-Gly-Asp peptide (Fig. 3 D). As seen in macrophages, inhibition of the α₅β₁-induced increase in CamKII activity by α_vβ₃ ligation was blocked by KN62, but not KN04.

Previously we have demonstrated that the cytoplasmic tail of β₃ is both necessary and sufficient for α_vβ₃ inhibitory crosstalk to α₅β₁ (reference 3 and Fig. 2, A and B). In the presence of mAb-16 beads, expression of Tacβ₃, but not Tacβ₅, prevented the α₅β₁-mediated rise in CamKII activity (Fig. 3 C). These results indicate that α₅β₁ and α_vβ₃ differentially regulate CamKII activity in macrophages and K562 cells.

To determine if the failure of the Kα_vβ₃S752A to initiate crosstalk was related to an inability to regulate CamKII, we evaluated CamKII activity after mAb-16 bead binding in K562 cells transfected with wild-type α_vβ₃ and the S752A and Y747F mutants. While ligation of α_vβ₃ with the β₃-specific mAb 7G2 suppressed mAb-16 bead-induced activation of CamKII, mutation of Ser752 of β₃ prevented the suppression of CamKII activity seen upon α_vβ₃ ligation (Fig. 4 A). However, mutation of Tyr747 or Tyr759 (data not shown) did not affect α_vβ₃ regulation of CamKII. Thus, Ser752 is required for both α_vβ₃ inhibitory crosstalk to α₅β₁ (Fig. 2) and α_vβ₃ regulation of CamKII (Fig. 4).

To determine the effect of α_vβ₃ and mutant β₃ on expression of CamKII, immunoprecipitates of CamKII were analyzed by Western blot with CamKII-specific Ab. As shown in Fig. 4 B, cellular expression of CamKII (see arrow) was unchanged by the expression of α_vβ₃ and mutants in transfected K562 cells.

Suppression of the α₅β₁-dependent increase in CamKII activity by α_vβ₃ ligation or by Tacβ₃ expression suggested that CamKII regulation could have a role in α_vβ₃ crosstalk to α₅β₁. To determine the role of CamKII in α_vβ₃ crosstalk to α₅β₁, Kα_vβ₃ cells were incubated with the α₅β₁ phagocytosis target, mAb-16 beads, or control target, P1F6 (anti-α_vβ₅) beads. Phagocytosis was measured in the presence and absence of 7G2 to ligate α_vβ₃, the CamKII inhibitor KN62, or control KN04. As reported previously, phagocytosis via α₅β₁ was inhibited upon α_vβ₃ ligation with mAb 7G2 (2, 3). α₅β₁ phagocytosis also was inhibited by KN62 (Fig. 5 A), but not KN04. Combining 7G2 and KN62 resulted in no further decrease in α₅β₁ phagocytosis. Under all conditions, there was no significant internalization of P1F6 beads.

To determine the dependence of α₅β₁ phagocytosis on CamKII activation, we evaluated phagocytosis in untransfected K562 cells which express α₅β₁, but not α_vβ₃. The absence of α_vβ₃ in these cells permitted the use of FN-coated beads as a phagocytosis target for α₅β₁ rather than the more selective mAb-16 beads used when α_vβ₃ is present. K562 phagocytosis of FN-coated beads via α₅β₁ was inhibited by the CamKII inhibitor KN62 (Fig. 5 B), but not the control KN04. Thus, enhanced CamKII activity, initiated by α₅β₁ binding of mAb-16 beads, appears to be required for α₅β₁ phagocytosis.

These data support the hypothesis that ligation of α₅β₁ stimulates CamKII activity and that α_vβ₃-mediated suppression of this activity is at least in part responsible for its inhibition of α₅β₁-mediated phagocytosis. To demonstrate that a similar mechanism was responsible for the inhibitory β₃ crosstalk to α₅β₁ during migration, we evaluated the effects of the CamKII inhibitor KN62 on K562 cell migration in response to FN. KN62, but not the inactive analogue KN04, inhibited the FN-induced migration of mock transfected K562 cells and KTacβ₅ (Fig. 5 C). The presence of KN62 did not further attenuate the minimal migration of Kα_vβ₃ or KTacβ₃ cells.

To test the hypothesis that α_vβ₃ crosstalk to α₅β₁ was a result of CamKII downregulation by β₃, K562 cells were infected with an adenovirus-directing expression of a constitutively active form of CamKII (1). Expression of this construct in untransfected K562 cells resulted in an eightfold increase in CamKII activity over a control viral construct encoding β-galactosidase (Fig. 6 A).

Next, KTacβ₃ and KTacβ₅ infected with virus encoding either β-galactosidase or constitutively active CamKII were assayed for their ability to migrate in response to FN. Expression of the active kinase specifically overcame the constitutive inhibition of α₅β₁-mediated migration in KTacβ₃, without any effect on migration in KTacβ₅ (Fig. 6 B). Thus, expression of active CamKII overcame α_vβ₃-mediated suppression of α₅β₁ high affinity functions. Unfortunately, safety concerns precluded testing the effect of the constitutively active CamKII in the phagocytosis assay.

Integrin crosstalk is an important mechanism for coordinating signals from multiple simultaneously ligated integrins on a single cell for a functional response to extracellular matrix. Although sometimes called “transdominant inhibition,” crosstalk may induce, as well as suppress functions of the target integrin, so we believe the more general term, preferable (9). Although the number of examples of integrin crosstalk has rapidly expanded in the past few years, little is known concerning the molecular mechanisms by which one integrin affects the function of another. K562 cells have proved a valuable model for examination of integrin crosstalk because these cells express a single integrin, α₅β₁, permitting a wide variety of genetic experiments exploring the basis of integrin crosstalk. In this system, we have previously shown that ligation of transfected α_vβ₃ inhibits the high affinity phagocytic function of α₅β₁ without effect upon low affinity α₅β₁-mediated adhesion and that the β₃ cytoplasmic tail is both necessary and sufficient for this effect. We now have used this model to explore the biochemical mechanisms involved in crosstalk. Based on a previous report, we examined a potential role for CamKII in α_vβ₃-mediated suppression of the high affinity functions of α₅β₁, and performed structure-function analysis of the β₃ cytoplasmic tail to further delineate the required structures for this unique signaling event.

In this study, we show that either α_vβ₃ ligation or expression of the isolated β₃ cytoplasmic tail exerts an inhibitory effect upon α₅β₁-mediated migration as well as phagocytosis. Since both α₅β₁ migration and phagocytosis are events that require the high affinity state of the integrin, and since the α_vβ₃-mediated inhibition of α₅β₁ is reversed by KN62 in both cases, these data suggest that a common signaling mechanism is responsible for these crosstalk events.

Based on the data in this report, we propose the hypothesis that CamKII, a ser/thr kinase with multiple intracellular substrates, is an important regulator of α₅β₁ function and a target of integrin crosstalk. First, ligation of α₅β₁ by specific antibody- or ligand-coated beads enhances the activity of CamKII in both macrophages and K562 cells. Second, activation of CamKII by ligation of α₅β₁ is required for both phagocytosis and migration. In contrast CamKII inhibitors do not affect adhesion which can be effected by low affinity α₅β₁. Thus, the requirement for CamKII activation appears to be specific for the high affinity functions of α₅β₁.

Coligation of α_vβ₃, or exposure of the isolated β₃ cytoplasmic tail, prevents α₅β₁-induced rise in CamKII activity. Since the β₃ integrin and the CamKII inhibitor have the same effect on α₅β₁ function, the data suggest that suppression of the ability of α₅β₁ to activate CamKII may be an important mechanism of integrin crosstalk. A role for CamKII suppression in integrin crosstalk is supported by the reversal of crosstalk inhibition of migration with constitutively active CamKII. Thus, our data support the hypothesis that α₅β₁-mediated CamKII activation is required for the high affinity functions of migration and phagocytosis and that α_vβ₃-activated crosstalk suppresses these functions through inhibition of CamKII activation. Thus, α₅β₁ and α_vβ₃ have opposing effects on CamKII activity.

Neither the upstream events regulating CamKII nor its downstream effector are yet known. Tyrosine kinase inhibitors have no effect either on the high-affinity functions of α₅β₁ or on suppression by α_vβ₃, suggesting the possibility that the entire pathway is independent of the well-known effects of integrin ligation on several tyrosine kinases (2, 3). Indeed, the independence of integrin crosstalk from the phosphorylation of Tyr747 further suggests that the signaling involved in the regulation of CamKII may be completely independent of these pathways. A recent report by Wu et al. (25) demonstrates that ligation of α₅β₁ and α_vβ₃ have opposite effects on plasma membrane calcium channel activity. Since calcium is an important regulator of CamKII, this voltage gated calcium channel may be important in the differential regulation of CamKII by these two integrins. Based on our preliminary pharmacologic data, one likely effector for CamKII in α₅β₁ high affinity function is myosin light chain kinase (MLCK). MLCK inhibitors KT5926 and ML9 both reverse α_vβ₃ inhibition of α₅β₁-mediated phagocytosis and migration without affecting inhibition of CamKII activation by α_vβ₃ ligation (Blystone, S.D., and E.J. Brown, unpublished data). MLCK phosphorylation by CamKII is known to inhibit MLCK activity, leading presumably to decreased myosin-induced cell traction (21). This integrin-mediated modulation of myosin function is consistent with the known role for myosin in phagocytosis and migration.

Analysis of structural requirements in the β₃ cytoplasmic tail in α_vβ₃-mediated crosstalk reveals that Ser752 of the β₃ cytoplasmic tail is required for inhibition of CamKII and for initiation of integrin crosstalk, while crosstalk is independent of either of the β₃ cytoplasmic tail tyrosines. The requirement for β₃ Ser752 in crosstalk is unexpected. The importance of Ser752 was suggested by a mutation to proline in a patient with Glanzmann's Thrombasthenia which abolished high affinity binding of fibrinogen by platelet α_IIbβ₃ (6, 8). However, detailed analysis has shown that mutation of Ser752 to Ala does not affect ligand binding by α_IIbβ₃ (8). The failure of the Ser752 to Ala β₃ mutation to affect ligand binding is supported by our studies in K562 which demonstrate normal adhesion, normal migration (Fig. 2, B and C), and normal generation of the ligand-induced binding site (LIBS) recognized by the antibody LIBS-1 in response to RGD peptide in this mutant (data not shown). In contrast, integrin crosstalk is entirely abolished by the S752A mutation, as it is by mutation to Pro (the original Glanzmann's mutation), to Glu (to mimic a potential phosphorylation), and to Cys (as a conservative mutation). Thus, it appears that Ser is absolutely required at this position. While this suggests the possibility of Ser phosphorylation in integrin crosstalk, we have been unable to demonstrate such phosphorylation so far. In contrast, Tyr747, which is absolutely required for stimulated adhesion and for α_vβ₃-mediated migration (Fig. 1) in K562 cells, is not involved in integrin crosstalk. Thus, these two amino acids, closely spaced in the relatively short cytoplasmic domain of one chain of an integrin, mediate two entirely distinct signaling cascades.

In a recent report, Bouvard et al. (5) showed that increased CamKII levels resulted in a decrease in the affinity of α₅β₁ for FN. In this in vitro system, CamKII and the phosphatase calcineurin regulate α₅β₁ affinity. Because the β₃ suppression of α₅β₁ phagocytosis occurs subsequent to α₅β₁ binding of ligand (3), it is possible that repeated cycling of α₅β₁ affinity is required for phagocytosis and migration. Binding of ligand-coated beads to high affinity α₅β₁ would activate CamKII, which would then decrease integrin affinity. This hypothesis predicts that integrin crosstalk from α_vβ₃ which blocks CamKII activation would prevent α₅β₁ movement to the low affinity state. This is entirely consistent with reports of receptor activation rather than inactivation by integrin crosstalk (14, 24) which measured ligand binding rather than functions that require affinity modulation.

Finally, these data demonstrate that, while increased CamKII activity is required for α₅β₁-mediated phagocytosis and migration, α_vβ₃ can perform these same functions independent of any increase in CamKII. This is a startling example of the diversity of signaling and function among the integrins. It suggests that there may be fundamental differences within this family of closely related receptors in how they mediate even their most basic functions. While many studies have emphasized common features of integrin α- and β-chains in association with cytoskeleton, calreticulin, and signaling molecules, the differences between α_vβ₃ and α₅β₁ in requirements for phagocytosis and migration suggest that there will be profound differences among integrins even as they perform similar functions.

The authors wish to thank all contributors of cDNAs and antibodies used in these studies and the members of the Brown laboratory for suggestions and advice on the performance of these studies.

CamKII

calcium/calmodulin-dependent protein kinase II

FN

fibronectin

IMDM

Iscove's Modified Eagle's Medium

LIBS

ligand-induced binding site

MLCK

myosin light chain kinase

VN

vitronectin